Methods of monitoring HIV drug resistance

ABSTRACT

a method is provided for detecting a presence of HIV virus in a sample comprising: taking a culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors necessary to allow the HIV virus to infect, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the cell culture with a sample to be analyzed for the presence of HIV virus in the sample; and detecting a change in a level of expression of the reporter gene in cells in the recombinant cell culture. The method can be used to detect the presence of HIV virus in a sample, detect the presence of different strains of HIV virus in a sample, detect HIV drug resistance in a sample, determine what combination of one or more anti-HIV agents would be effective in treating a patient, and screen compositions for anti-HIV activity.

RELATIONSHIP TO COPENDING APPLICATIONS

[0001] This application claims priority to “METHODS OF MONITORING HIVDRUG RESISTANCE,” Provisional Application Serial No.: 60/117,136; Filed:Jan. 25, 1999 which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to recombinant cell lines andmethods for detecting and monitoring viral infection. More particularly,the invention relates to recombinant cell lines and methods fordetecting HIV infection, monitoring HIV for drug resistance andscreening for anti-HIV agents.

[0004] 2. Description of Related Art

[0005] Human immunodeficiency virus (HIV) has been implicated as theprimary cause of the slowly degenerate disease of the immune systemtermed acquired immune deficiency syndrome (AIDS). Infection of the CD4⁺subclass of T-lymphocytes with the HIV type-1 virus (HIV-1) leads todepletion of this essential lymphocyte subclass which inevitably leadsto opportunistic infections, neurological disease, neoplastic growth andeventual death.

[0006] Infection with human immunodeficiency virus (HIV) is a chronicprocess with persistent, high rates of viral replication. Thepathogenesis of HIV-1 infection is characterized by a variable but oftenprolonged asymptomic period following the acute viremic phase. Previouswork has established a correlation between HIV disease progression andincreasing amounts of infectious virus, viral antigens, andvirus-specific nucleic acids (Ho et al., New England. J. Med. 321:1621-1625 (1989); Schnittman et al. AIDS Res. Hum. Retroviruses 7:361-367 (1991); Pantalco et al. Nature 362: 355-358 (1993)).

[0007] A variety of reagents and assays have been developed to detectthe infection of HIV and monitor the progression of HIV in the body. Forexample, counting the depletion of CD4⁺ cells has been used to indicatethe prognosis of AIDS. Serological screening techniques are also beingutilized worldwide for the detection of HIV, where the presence of theantibody against HIV antigens, such as the HIV p24 antigen, is detected.

[0008] An ELISA assay is currently being utilized on serum samples inmost hospitals and screening laboratories to make the determination.However, currently used ELISA assays may not be sensitive enough todetect all HIV infected individuals. This is because that some HIVinfected individuals do not have detectable levels of serum antibody toHIV. There may be a significant time lag between detection of HIVinfection and seroconversion. In addition, some HIV infected butseronegative individuals might never convert but will remain infectedthroughout theirs lives. Thus, such a screening method may generatefalse negatives, which in turn may increases the probability of HIVinfection of healthy people by these individuals.

[0009] Another method for detecting HIV infection in seronegativeindividuals was described (Jehuda-Cohen, T. et al. Proc. Natl. Acad.Sci. UAS, 87: 3972-3076 (1990)) wherein peripheral blood mononuclearcells (PBMC) are isolated from the blood and then exposed to a mitogensuch as pokeweed mitogen. Incubation of isolated PBMC with pokeweedmitogen caused the PBMC to secret immunoglobulins that were specific forHIV. The failure of the ELISA assay to detect all HIV infectedindividuals places the population at risk by misleading the HIV infectedindividuals that they are not infected, thereby making it more likelythat the HIV infected individuals will unknowingly infect others.

[0010] The existence of HIV has also been determined by using thereverse transcriptase-polymerase chain reaction (RT-PCR) to amplifyplasma HIV RNAs (U.S. Pat. No. 5,674,680). This method is used to detectthree types of HIV mRNA in peripheral blood cells: unspliced, multiplespliced, and single-spliced mRNA in AIDS patients, HIV-infected butasymptomatic individuals and individuals who are undergoing therapy forAIDS. However, the correlation between the differences in HIV mRNAlevels and AIDS prognosis needs to be established.

[0011] Many antiviral drugs have been developed to inhibit HIV infectionand replication by targeting HIV reverse transcriptase and proteases.Treatment following a prolonged single drug regimen has met with limitedsuccess where there is relatively small drop in viral load, followed bya rise in amount of detectable virus in blood, presumably due to thedevelopment of drug resistance strains of HIV. The resistance of HIV todrugs is not only associated with the high mutation rates of HIV butalso due to the selective pressure of prolonged anti-HIV drug therapy.Since the original description of diminished susceptibility of isolatesof HIV-1 to zidovudine (AZT) (Larder et al. Science (1989)243:1731-1734), the literature has disclosed many descriptions ofdiminished susceptibility to AZT in different clinical situations, withdifferent assay systems, and of genetic mutations responsible forchanges in susceptibility. For example, isolates from subjects nottreated with AZT display a narrow range of susceptibilities to AZT, withthe 50% inhibitory concentrations (IC50) ranging from 0.001 to 0.04 μM(Larder et al. (1989), supra; Rooke et al. AIDS (1989) 3:411-415; Landet al. J Infect Dis (1990) 161:326-329; Richman et al. J. AIDS (1990)3:743-746; Tudor-Williams et al. Lancet (1992) 339:15-19). This narrowrange of susceptibilities is typical for HIV isolates from subjects ofall ages and at all stages of HIV infection. Isolates of HIV frompatients who receive AZT, however, chronically display progressivereductions of susceptibility to AZT over periods of months to years.Diminished susceptibility to AZT of an isolate of HIV-2 from a patienton prolonged therapy has also been reported (Pepin et al. EighthInternational Conference on AIDS, Amsterdam, The Netherlands, Jul.19-24, 1992 Abstract PoA 24401).

[0012] In addition to AZT, HIV resistance have been seen with othernucleosides and to nonnucleoside anti-retroviral drugs. For example,isolates resistant to AZT display diminished susceptibility to othernucleosides containing a 3′-azido moiety, including3′-azido-2′,3′-dideoxyuridine, 3′-azido-2′,′dideoxyguanosine, and3′-azido-2′,3′-dideoxyadenosine (Larder et al. (1989), supra; Larder etal. Antimicrob Agents Chemother (1990) 34:436-441). Additionally,AZT-resistant isolates are reported to display cross-resistance todidehydrodideoxythymidine (Rooke et al. Antimicrob. Agents Chemother.(1991) 35:988-991).

[0013] Drug resistance in HIV isolates is not limited to inhibitors ofreverse transcriptase and virtually all drug targets for anti-HIVtherapy are susceptible to the development of resistance. For example, amutant with resistance to a protease inhibitor has been isolated thatexhibits an eightfold reduction in susceptibility to a proteaseinhibitor (Patterson et al. Eighth International Conference on AIDS,Amsterdam, The Netherlands, Jul. 19-24, 1992, Abstract ThA 1506).

[0014] In the last five year, with the fast development of anti-HIVdrugs and utilization of combination therapy, treatment of HIV infectionwith multiple antiviral drugs (“cocktails”) have led to diminutions inthe amount of viral RNA and virus detectable in blood by using currentdetection methods. It has been shown that combination therapy with 3 ormore antiviral drugs, e.g. indinavir, zidovudine, and lamivudine, oralternatively, nevirapine, zidovudine, and didanosine, in previouslyuntreated patients has resulted in profound decreases in viral burden(Wainberg, M. A. and Friedland, G. JAMA (1998) 279: 1977-1983). It wasbelieved that the combination antiviral regimens used must have blockedviral replication to the extent that the mutations that encode drugresistance could not occur. However, current studies showed that agrowing number of patients are failing combination drug regimens (Deek,S. et al. the 5th Conference on Retroviruses and OpportunisticInfection, Chicago, Feb. 1-5, 1998, Abstract #419). Finding an effectivesalvage therapy for them is difficult.

[0015] In the clinical setting, drug resistance is often not detecteduntil a patient manifests symptoms of disease progression, which isgenerally not observed until significantly after development of a drugresistant strain of virus. Thus, there is a clear need for an assaywhich can indicate the drug resistance of virus strains so drug therapyfor a patient can be modified accordingly, and optimally as soon asresistance is detected rather than delaying until clinical symptoms areobserved.

[0016] Currently the most commonly used assays for susceptibility of HIVto antiviral drugs involve the measurement of the inhibition ofcytopathology, p24 production, or reverse transcriptase production of alaboratory strain of HIV in a lymphoblastoid cell line. Such assays maynot be readily applied to clinical isolates of HIV. Examples of commonlyused assays of drug susceptibility of clinical isolates have been thesyncytial focus assay in CD4-HeLa cells (Chesebro, B. and Wehrly, K., J.Virol. (1988) 62:3779-3788), inhibition of p24 production in primaryperipheral blood mononuclear cells, and reverse transcriptase (RT)assays using cultured primary T-cells from patient blood. (Richman etal. In: Current Protocols in Immunology, Coligan et al., eds, (1993)Brooklyn, J. Wiley). .

[0017] One of the disadvantages associated with the syncytial focusassay is that it may only detect HIV viruses that exhibit asyncytial-inducing phenotype and that in practice may only be obtainedfrom a minority of specimens from seropositive individuals. And thesyncytial focus assays may not be used for screening for drugs thataffect posttranslational processing, such as glycosidase and proteaseinhibitors. On the other hand, the p24 and RT assays may also suffer thelimitations of difficult quantitation, low sensitivity and unprovenclinical validity.

SUMMARY OF THE INVENTION

[0018] A recombinant cell is provided which comprises: a reportersequence introduced into the recombinant cell comprising a reporter genewhose expression is regulated by a protein specific to HIV viruses whichis expressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; the recombinant cell being capable ofcell division and expressing a CD4 receptor and one or more additionalcell surface receptors which facilitate productive infection of therecombinant cell by the HIV virus; and the recombinant cell enabling HIVvirus which has infected the recombinant cell to replicate and infectnon-infected cells in a culture of the recombinant cell.

[0019] As used herein, introducing a reporter sequence into arecombinant cell refers to the introduction of a sequence into cell byany of a variety of recombinant methodologies including, but not limitedto, transformation, transfection and transduction.

[0020] The recombinant cell may optionally express a sufficient numberof cell surface receptors to render the recombinant cell permissive tosubstantially all strains of HIV. Alternatively, the recombinant cellmay express a selected group of cell surface receptors such that therecombinant cell is permissive to a selected group of strains of HIV.Examples of cell surface receptors which may be expressed by therecombinant cell include, but are not limited to CXCR4, CCR5, CCR1,CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1, STRL33/BONZO andGPR15/BOB.

[0021] The stably transferred reporter sequence may optionally comprisea promoter sequence including an HIV virus specific enhancer sequence,and a reporter gene whose expression is regulated by binding of an HIVspecific transactivator protein to the HIV specific enhancer sequence.According to this variation, the HIV specific transactivator protein ispreferably Tat and the HIV specific enhancer sequence preferablycomprises at least one copy of TAR sequence. Alternatively, the HIVspecific protein may optionally regulates expression of the reportersequence by a protein-protein interaction between the HIV specificprotein and a transactivator protein present in the recombinant cell.

[0022] Examples of the HIV specific protein include, but are not limitedto, HIV proteins Tat, Rev, Vpr, Vpx, Vif, Vpu, Nef, Gag, Env, RT, PR,and IN. The HIV specific protein may optionally be an HIV transactivatorprotein such as Tat.

[0023] Expression of the reporter gene in the recombinant cell may be isup-regulated or down-regulated by the HIV specific protein.

[0024] A method is provided for detecting a presence of HIV virus in asample comprising: taking a culture of recombinant cells which (a) arecapable of cell division, (b) express CD4 receptor and one or moreadditional cell surface receptors necessary to allow the HIV virus toinfect, (c) enable the HIV virus to replicate and infect the noninfectedcells in the cell culture, and (d) comprise a reporter sequenceintroduced into the recombinant cells comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; contacting the cell culture with asample to be analyzed for the presence of HIV virus in the sample; anddetecting a change in a level of expression of the reporter gene incells in the recombinant cell culture.

[0025] A method is also provided for detecting the presence of differentstrains of HIV virus in a sample comprising: taking a first culture ofrecombinant cells which (a) are capable of cell division, (b) expressCD4 receptor and one or more additional cell surface receptors whichrender the first cell culture permissive to a first group of strains ofHIV but does not render the first cell culture permissive to a second,different group of strains of HIV, (c) enable the HIV virus to replicateand infect the noninfected cells in the cell culture, and (d) comprise areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus; taking a second culture ofrecombinant cells which (a) are capable of cell division, (b) expressCD4 receptor and one or more additional cell surface receptors whichrender the second culture permissive to the second group of strains ofHIV but does not render the second cell culture permissive to the firstgroup of strains of HIV, (c) enable the HIV virus to replicate andinfect the noninfected cells in the cell culture, and (d) comprise areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus; contacting the first andsecond cell cultures with a sample to be analyzed for the presence ofdifferent strains of HIV virus; detecting a change in a level ofexpression of the reporter gene in cells in the first cell culture;detecting a change in a level of expression of the reporter gene incells in the second cell culture; and distinguishing between the firstand second groups of strains based on whether a change in a level ofexpression of the reporter gene occurs in the first or the second cellculture.

[0026] According to the above method, the first and second cultures ofrecombinant cells may optionally be mixed with each other. The reportergenes in the first and second cultures of recombinant cells may alsooptionally be different from each other so that cells of the first cellculture can be distinguished from cells of the second cell culture. Thisallows different strains of HIV virus to be detected in a single wellcontaining cells from both cultures.

[0027] A method is also provided for detecting HIV drug resistance in asample comprising: taking a culture of recombinant cells which (a) arecapable of cell division, (b) express CD4 receptor and one or moreadditional cell surface receptors necessary to allow the HIV virus toinfect, (c) enable the HIV virus to replicate and infect the noninfectedcells in the cell culture, and (d) comprise a reporter sequenceintroduced into the recombinant cells comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; contacting the cell culture with asample containing HIV virus; adding one or more anti-HIV agents to thecell culture either before or after contacting the cell culture with thesample; and detecting a change in a level of expression of the reportergene in the cells.

[0028] A method is also provided for taking a patient known to beinfected with one or more strains of the HIV virus and determining whatcombination of one or more anti-HIV agents would be effective intreating the patient, the method comprising: taking a plurality of cellcultures, each of the cultures containing recombinant cells which (a)are capable of cell division, (b) express CD4 receptor and one or moreadditional cell surface receptors necessary to allow the HIV virus toinfect, (c) enable the HIV virus to replicate and infect the noninfectedcells in the cell culture, and (d) comprise a reporter sequenceintroduced into the recombinant cells comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; contacting the cell cultures with asample containing the HIV virus; adding a different set of one or moreanti-HIV agents to each of the cell cultures, either before or aftercontacting the cell cultures with the sample; and comparing expressionof the reporter gene in the plurality of cell cultures.

[0029] A method for screening compositions for anti-HIV activitycomprising: taking a culture of recombinant cells which (a) are capableof cell division, (b) express CD4 receptor and one or more additionalcell surface receptors necessary to allow the HIV virus to infect, (c)enable the HIV virus to replicate and infect the noninfected cells inthe cell culture, and (d) comprise a reporter sequence introduced, intothe recombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the cell culture with a sample containing the HIVvirus; adding one or more agents whose anti-HIV activity are unknown tothe cell culture, either before or after contacting the cell cultureswith the sample; and detecting a change in a level of expression of thereporter gene in the cells in the culture.

[0030] According to any one of the above methods, the recombinant cellsin the cell cultures used in the methods may optionally comprise areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus; the recombinant cells beingcapable of cell division and expressing a CD4 receptor and one or moreadditional cell surface receptors which facilitate productive infectionof the recombinant cell by the HIV virus; and the recombinant cellsenabling the HIV virus which has infected the recombinant cell toreplicate and infect non-infected cells in a culture of the recombinantcell.

[0031] Also according to any one of the above methods, the HIV specificprotein may be any one of the HIV proteins Tat, Rev, Vpr, Vpx, Vif, Vpu,Nef, Gag, Env, RT, PR, and IN. The HIV specific protein may optionallybe an HIV transactivator protein such as Tat.

[0032] Also according to any one of the above methods, the reportersequence may comprise a promoter sequence including an HIV virusspecific enhancer sequence, and a reporter gene whose expression isregulated by binding of an HIV specific transactivator protein to theHIV specific enhancer sequence. In one variation, the HIV specifictransactivator protein is Tat and the HIV specific enhancer sequencecomprises at least one copy of TAR sequence.

[0033] Also according to any one of the above methods, the one or moreadditional cell surface receptors expressed by the recombinant cell mayinclude, but are not limited to CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4,CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1, STRL33/BONZO and GPR15/BOB.

[0034] Also according to any one of the above methods, detecting achange in a level of expression of the reporter gene in the cells mayinclude detecting a change in a level of expression of the reporter genein individual cells.

[0035] Also according to any one of the above methods, detecting achange in a level of expression of the reporter gene in the cells mayinclude detecting a change in a level of expression of the reporter geneacross the cell culture.

[0036] Also according to any one of the above methods, detecting achange in a level of expression of the reporter gene in the cells mayinclude detecting whether viral replication within the cell culture hasoccurred.

[0037] Also according to any one of the above methods, detecting achange in a level of expression of the reporter gene in the cells mayinclude comparing a level of expression in cells contacted with thesample to a level of expression cells contacted with one or more controlsamples.

[0038] Also according to any one of the above methods, the sample may beany sample which might include HIV including, but not limited to wholeblood, blood serum, isolated peripheral blood cells,T cells, and bonemarrow.

[0039] Kits are also provided for performing the various methods of thepresent invention. These kits may include the cell line of the presentinvention and any two or more components used to perform these methods.

[0040] In one variation, a kit is provided which comprises: first andsecond recombinant cell lines, each recombinant cell line comprising: areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus, the recombinant cell linebeing capable of cell division and expressing a CD4 receptor and one ormore additional cell surface receptors which facilitate productiveinfection of the recombinant cell by the HIV virus, and the recombinantcell line enabling the HIV virus which has infected the recombinant cellto replicate and infect non-infected cells in a culture of therecombinant cell; wherein the one or more additional cell surfacereceptors which the first recombinant cell line expresses renders thefirst recombinant cell line permissive to a first group of strains ofHIV and the one or more additional cell surface receptors which thesecond recombinant cell line expresses renders the second recombinantcell line permissive to a second, different group of strains of HIV.

[0041] According to this variation, the first and second recombinantcell lines may optionally be mixed together in the kit. Also accordingto this variation, the first recombinant cell line may optionallyinclude a first reporter gene and the second recombinant cell line mayoptionally include a second different reporter gene which allows thefirst and second recombinant cell lines to be independently identified.

BRIEF DESCRIPTION OF THE FIGURES

[0042]FIG. 1A illustrates expression plasmids for HIV receptors and areporter gene.

[0043]FIG. 1B illustrates retroviral vectors for HIV receptors and areporter gene.

[0044]FIG. 2A illustrates an expression plasmid for human CD4 and CXCR4receptors.

[0045]FIG. 2B illustrates a plasmid for a lacZ reporter gene.

[0046]FIG. 3A shows HeLaT4 cells cultured in the presence of HIV virusand later processed with X-Gal.

[0047]FIG. 3B shows HeLa D4R4 cells cultured in the presence of HIVvirus and later processed with X-Gal 1 day after the initial infection.

[0048]FIG. 3C shows HeLa D4R4 cells cultured in the presence of HIVvirus and later processed with X-Gal 3 days after the initial infection.

[0049]FIG. 3D shows HeLa D4R4 cells cultured in the presence of HIVvirus and later processed with X-Gal 4 days after the initial infection.

[0050]FIG. 3E shows HeLa D4R4 cells cultured in the presence of HIVvirus and later processed with X-Gal 5 days after the initial infection.

[0051]FIG. 3F shows HeLa D4R4 cells cultured in the presence of HIVvirus and AZT and later processed with X-Gal.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention relates to new and useful methods includingmethods for detecting HIV, methods for detecting HIV drug resistance,methods for designing patient customized anti-HIV drug cocktailtreatments, and methods for screening compositions for anti-HIVactivity. Also provided are novel cell lines which may be used with themethods of the present invention.

[0053] The methods of the present invention use cells which (a) arecapable of cell division; (b) are permissive to HIV virus; (c) express areporter gene whose expression is selectively regulated by infectionwith HIV; and (d) allow viral replication of HIV in infected cells whichenables cells within the same cell culture which are initiallyuninfected to become infected.

[0054] One of the advantages provided by the present invention is thatthe recombinant cells used are capable of cell division. As a result, itis easy to produce and maintain these cells for performing the variousmethods of the present invention.

[0055] A further advantage provided by the present invention is that therecombinant cells can be infected by multiple different strains of HIV,including wild-type and mutant HIV strains from clinical isolates orlaboratory-adapted strains. As a result, the methods of the presentinvention have broad applicability to all strains of HIV.

[0056] Yet a further advantage provided by the present invention is thatinfection of the recombinant cells by an HIV virus can be easilymonitored and measured. By using a reporter gene whose expression isregulated by infection with HIV, it is possible to detect HIV infectionby simple detection methods, such as calorimetric methods. By expressionof the reporter gene being selectively regulated by infection with HIV,false positive signals, for example due to infection by non-HIV viruses,are reduced.

[0057] A further advantage of the present invention is that therecombinant cells not only allow entry and infection of the HIV virus,but also facilitate efficient replication within the recombinant celland transmission of the mature HIV virion to infect other cells in theculture. By using a cell line in which HIV is able to infect some cellsin a cell culture, replicate, and then infect other cells in the cellculture, as well as by coupling viral replication with cell division,the signal produced by the reporter gene is amplified since more cellsare infected than would be infected absent replication of HIV within thecell culture. For example, a single virion contained in a sample isultimately able to infect all cells in the cell culture. This featureallows for sensitive detection of the HIV virus contained in a samplethat is applied to the recombinant cell culture.

[0058] By exploiting the above-described advantages, as well as featuresfurther described in details below, the recombinant cell line can beused in a variety of methods or assays for many laboratory and clinicalapplications relating to HIV.

[0059] It should be noted that the methods and cells of the presentinvention can be modified and adapted for various viruses other thanHIV, including but are not limited to retroviruses, coronaviruses,herpes viruses and adenoviruses. For example, an immortalized cell linecan be constructed to comprise a panel of receptors and coreceptors toallow infection, replication and amplification of one or more strains ofa target virus; and a reporter gene whose expression is regulated by aspecific gene product expressed by the target virus.

[0060] 1. Recombinant Cell Line

[0061] One aspect of the present invention relates to recombinant cellsfor use in detecting infection by an HIV virus. In one embodiment, therecombinant cell comprises:

[0062] a reporter sequence introduced into the recombinant cellscomprising a reporter gene whose expression is regulated by a proteinspecific to HIV viruses which is expressed from a genome of an HIV virusupon infection of the recombinant cell by the HIV virus;

[0063] the recombinant cell being capable of cell division andexpressing a CD4 receptor and one or more additional cell surfacereceptors which facilitate productive infection of the recombinant cellby the HIV virus; and

[0064] the recombinant cell enabling the HIV virus which has infectedthe recombinant cell to replicate and infect non-infected cells in aculture of the recombinant cell.

[0065] Regulation of the reporter gene expression may involveup-regulation where the HIV specific protein causes expression of thereporter gene to begin or to increase. Alternatively, regulation of thereporter gene expression may involve down-regulation where the HIVspecific protein causes expression of the reporter gene to cease or todecrease.

[0066] The HIV specific protein may be an HIV transactivator proteinssuch as Tat, an HIV regulatory protein such as Rev, HIV accessoryproteins such as Vpr, Vpx, Vif, Vpu and Nef, HIV structural proteinssuch as Gag and Env, or HIV enzymatic proteins such as RT (reversetranscriptase), PR (protease) and IN (integrase). The regulation of thereporter sequence may be achieved by using various methods known in theart. For example expression of the reporter sequence can be regulated bydirect binding of the transactivator protein Tat to an enhancer sequenceupstream comprising at least one copy of TAR sequence. Alternatively,expression of the reporter gene can be regulated via protein-proteininteraction between the HIV specific protein and an transactivatorprotein present in the recombinant cell.

[0067] In one variation of this embodiment, the reporter sequence in therecombinant cell comprises a promoter sequence including an HIV virusspecific enhancer sequence, and a reporter gene whose expression isregulated by binding of an HIV specific transactivator protein to theHIV specific enhancer sequence.

[0068] According to this preferred embodiment, regulation of thereporter gene expression in the recombinant cells is achieved by using apromoter sequence including an HIV virus specific enhancer sequencewhich is transcriptionally responsive to an HIV specific transactivatorprotein. Upon infection by the HIV virus, the HIV specifictransactivator protein expressed from the HIV genome binds to the HIVspecific enhancer sequence and enhances expression of the reporter gene.The presence, absence or level of the reporter gene product is detectedand used to indicate the infection of the HIV virus.

[0069] In a particularly preferred variation, the reporter sequencecomprises at least one copy of TAR sequence as the HIV virus specificenhancer sequence. Expression of the reporter sequence is regulated bythe binding of the HIV specific transactivator protein Tat to theenhancer sequence TAR.

[0070] a wide variety of reporter genes may be used in the presentinvention, Examples of proteins encoded by reporter genes include, butare not limited to, easily assayed enzymes such as 0-galactosidase,luciferase, beta-glucuronidase, chloramphenicol acetyl transferase(CAT), secreted embryonic alkaline phosphatase (SEAP), fluorescentproteins such as green fluorescent protein (GFP), enhanced bluefluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP)and enhanced cyan fluorescent protein (ECFP); and proteins for whichimmunoassays are readily available such as hormones and cytokines. Theexpression of these reporter genes can also be monitored by measuringlevels of mRNA transcribed from these genes.

[0071] The one or more additional cell surface receptors expressed bythe recombinant cell may optionally include, but are not limited to,CXCR4, CCR5, other chemokine receptors such as CCR1, CCR2b, CCR3, CCR4,CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1, and chemokine receptor-like orphanproteins such as STRL33/BONZO and GPR15/BOB.

[0072] The presence of CD4 and these one or more additional cell surfacereceptors allows efficient entry, infection and replication of HIVstrains with different tropisms. By causing the recombinant cell toexpress as many cell surface receptors as possible, the recombinant cellmay be rendered permissive to virtually all strains of HIV, regardlessof tropism. This may be accomplished by transfecting or transducing thecell with all cell surface receptors known to be involved in HIVinfection or by cell fusion with cells, such as T-cells or monocytes,which express these receptors on the cell surface. Alternatively, bycausing the recombinant cell to express certain cell surface receptorsor sets of cell surface receptors, it is possible to design therecombinant cell to be permissive to certain strains of HIV and to notbe permissive to other strains of HIV. Thus, by selecting which cellsurface receptors are expressed, cell lines can be designed forscreening for particular strains or groups of strains of HIV virus.

[0073] The recombinant cell lines used in the present invention can beconstructed from a wide variety of immortalized cell lines. In oneembodiment, the recombinant cells are immortalized tumor cells. One ofthe advantages associated with using tumor cells is that tumor cellsundergo relatively fast cell cycling or division, which may furtherenhance replication and amplification of the virus in the culture. Theimmortalized tumor cell lines can be generated from primary tumor cellsor from established tumor cell lines. Alternatively, normal cells canalso be used so long as the cells are immortalized. Examples include butare not limited to primary cells immortalized by transfection withtelomerase gene and normal cells immortalized by SV40 tranformation.These immortalized cells can proliferate indefinitely, thus providing anample and economic supply of cells.

[0074] Compared to human T-cells that have been used in the art for HIVvirus production, the recombinant cell lines of the present inventionare relatively easier to culture, more stable, and less expensive. Ithas been acknowledged that the principle cell types targeted by HIV-1are helper T-lymphocytes and cells of the monocyte macrophage lineagevia the CD4 receptor pathway in vivo, while in tissue culture systems,HIV are cytopathic for CD4⁺-lymphocytes and cause dysfunction ofmacrophages, which is directly accounted for depletion of T cells in thebody. Since replicating HIV in infected individuals is readily detectedin peripheral blood and lymph lodes, human peripheral mononuclear cells(PBMC), in particular, have been frequently used as host cells for HIVinfection in vitro and anti-HIV drug-susceptibility testing. One of thedisadvantages with PBMC cells is that these primary cells have to beobtained from donors, carefully cultured and freshly prepared each time.It is costly and inefficient to use these primary T-cells for commercialpurposes. In addition, the permissiveness of these T-cells to differentstrains of HIV virus may vary with the donor, thus causing ambiguity inclinical testing. Thus, the recombinant cells of the present inventionwhich can be produced in an ample supply, are permissive to HIVinfection, relatively stable and can be cultured and manipulated moreeasily in vitro, are well suited for large scale commercial reproductionand use in high throughput screening.

[0075] 2. Methods for Detecting HIV in a Sample

[0076] Methods are provided for detecting a presence of HIV virus in asample. In one embodiment, the method comprises:

[0077] taking a culture of recombinant cells, which (a) are capable ofcell division, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in theculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus;

[0078] contacting the cell culture with a sample to be analyzed for thepresence of HIV virus in the sample; and

[0079] detecting a change in a level of expression of the reporter genein cells in the culture, such change being indicative of the HIV virusbeing present in the sample and infecting cells in the cell culture.

[0080] The culture of recombinant cells used in the method may be anycell culture which has the above described properties. The recombinantcells described in Section I are examples of cells having theseproperties and may be used in this method.

[0081] Detecting a change in a level of expression of the reporter genein the cells in the culture may be performed by detecting a change in alevel of expression of the reporter gene in individual cells or a changein a level of expression of the reporter gene across the cell culture.

[0082] In one embodiment, detecting a change in a level of expressionincludes detecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

[0083] In another embodiment, detecting a change in a level ofexpression includes comparing a level of expression in cells contactedwith the sample to a level of expression cells contacted with one ormore control samples. For example, cells contacted with a sample notcontaining HIV virus can serve as a negative control, while cellscontacted with a sample containing HIV virus, recombinant and stabilizedHIV virus, or another virus capable of infecting the cells and causingexpression of the HIV specific protein, such as a modified adenovirusencoding Tat, can serve as a positive control. By using suitablecontrols, induction of the reporter gene expression may be bettercorrelated with HIV infection.

[0084] It is noted that regulation of the reporter gene may be upregulation or down regulation. Accordingly, a change in the level ofexpression of the reporter gene may be an increase or decrease inreporter gene expression.

[0085] The methods described above can be used for diagnosis of HIVvirus contained in variety of samples including, but are not limited to,whole blood, blood serum, isolated peripheral blood cells, T cells,other biological fluids such as urine, saliva, tears and semen, as wellas isolated wild-type or mutant HIV viruses from laboratories orclinics. For example, whole blood of individuals can be tested for thepresence of HIV virus by using the methods described above. In addition,blood or bone marrow samples from individual donors or samples frompooled blood stored in blood banks can be screened for the presence ofHIV virus. The sensitivity of the methods to detect even a single HIVvirion allows for the diagnosis of HIV in individuals at a very earlystage of HIV infection and can be used to prevent HIV-positive bloodfrom being transfused into patients.

[0086] One advantage of using the above-described method for HIVdiagnosis is attributed to the specific response of the recombinantcells to HIV virus only. Because expression of the reporter gene isspecifically regulated by HIV specific gene products, ambiguity indiagnosis or report of false positives can be avoided in the clinic. Onthe other hand, by using the above-described method, HIV virus may bedetected in those individuals who are infected by HIV but do not havedetectable levels of serum antibody (seronegatives), thereby reducingthe incidents of false negatives which may arise from usingantibody-based detection methods.

[0087] The methods described above can also be used to amplify HIVvirus, especially strains with low occurrences in the blood sample andevasive to other detections. With the replication and amplification ofthe HIV virus in the recombinant cells, HIV virus with higher titer canbe generated in the cell culture and isolated for further studies suchas cloning of novel HIV strains.

[0088] The methods described above can also be used to differentiatestrains or tropisms of HIV viruses in a sample by using recombinantcells selectively expressing certain HIV coreceptors. For example, CXCR4coreceptor which is required by T-tropic strains can be selectivelyexpressed in a first recombinant cell line to allow infection ofT-tropic strains of HIV. Meanwhile, since M-tropic strains require CCR5coreceptor to infect cells, a second recombinant cell line can beconstructed to selectively express CCR5 to allow infection of M-tropicstrains of HIV. By having the first and second recombinant cell linesexpressing different coreceptors, the first and second recombinant celllines can selectively detect T-tropic, M-tropic or dual-tropic strainsin the presence of other strains of HIV virus.

[0089] Alternatively, the first recombinant cell line may include afirst reporter gene such as GFP, while the second recombinant cell linemay include a second reporter gene such as EBFP. When the first andsecond cell lines are mixed in one culture and contacted by a samplecontaining HIV virus with unknown tropism, selective expression of onereporter gene may indicate single tropism of the virus, while expressionof both reporter genes may indicate dual tropism. Differentfluorescences emitted by the first and second cell lines observed undermicroscope can facilitate independent identification of each cell linein one culture.

[0090] The methods described above can also be used for quantitativeanalysis of HIV virus in a sample. For example, by using control sampleswith varying titers, the viral load can be readily calculated bycomparing to the control samples. Alternatively, the viral titer of asample can also be determined by serially diluting the sample until endpoint infection is achieved in multiple cell culture plates, i.e. someof the cell culture plates are infected while the other plates are notinfected by the diluted sample.

[0091] 3. Methods for Detecting HIV Drug Resistance

[0092] Methods are also provided for detecting HIV drug resistance in asample. These methods may be used to detect whether a course oftreatment for HIV infection with one or more drugs is ineffective due tothe presence of one or more strains of HIV which are resistant to theone or more drugs being used. These methods may also be used to isolateHIV strains which are resistant to one or more anti-HIV agents.

[0093] In one embodiment, the method comprises:

[0094] taking a culture of recombinant cells, which (a) are capable ofcell division, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in theculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus;

[0095] contacting the cell culture with a sample containing HIV virus;

[0096] adding one or more anti-HIV agents to the cell culture eitherbefore or after contacting the cell culture with the sample; and

[0097] detecting a change in a level of expression of the reporter genein the cells.

[0098] Anti-HIV agents used in the methods may be any agents with knownanti-HIV activities, either tested preclinically or clinically. Examplesof anti-HIV agents which may be used to screen for HIV drug resistanceinclude, but are not limited to, nucleoside HIV RT inhibitors such asZIDOVUDINE, DIDANOSINE, ZALCITABINE, LAMIVUDINE, STAVUDINE, ABACAVIR,nonnucleoside RT inhibitors such as NEVIRAPINE, DELAVIRDINE, EFAVIRENZ,protease inhibitors such as INDINAVIR, RITONAVIR, SAQINAVIR, NELFINAVIR,AMPRENAVIR, and combinations thereof.

[0099] The culture of recombinant cells used in the method may be anycell which has the above described properties. The recombinant cellsdescribed in Section I are examples of cells having these properties andmay be used in this method.

[0100] Detecting a change in a level of expression of the reporter genein the cells in the culture may be performed by detecting a change in alevel of expression of the reporter gene in individual cells or a changein a level of expression of the reporter gene across the cell culture.

[0101] In one embodiment, detecting a change in a level of expressionincludes detecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

[0102] In another embodiment, detecting a change in a level ofexpression includes comparing a level of expression in cells contactedwith the sample to a level of expression cells contacted with one ormore control samples. For example, cells contacted with a samplecontaining HIV virus but not with the one or more anti-HIV agents canserve as a negative control, while cells contacted with a samplecontaining a HIV virus that is not known to be resistant to the one ormore anti-H IV agents added may preferably serve as a positive control.By using suitable controls, induction of the reporter gene expressionmay be better correlated with the resistance of the HIV virus to theagents.

[0103] It is noted that regulation of the reporter gene may be upregulation or down regulation. Accordingly, a change in the level ofexpression of the reporter gene may be an increase or decrease inreporter gene expression.

[0104] In one variation of this embodiment, the cell culture iscontacted with one or more anti-HIV drugs before being contacted with asample containing the HIV virus. Alternatively, the cell culture may becontacted with one or more anti-HIV drugs after being contacted with asample containing the HIV virus and incubating for a time sufficient forthe HIV virus replication to occur. This may be particularlyadvantageous for the initial amplification of the HIV virus with lowtiter in the sample before being tested for drug resistance.

[0105] The methods described above can be used to detect drug resistanceof HIV virus contained in patient samples, isolated virus stocks orlaboratory-adapted HIV strains. Owing to ultra sensitivity of therecombinant cells to a single HIV virion, the strains of HIV virus thatescape the drug regimen or the ones that are not predominant circulatingvariants can replicate in the cell culture and be isolated for furthergenotypical analysis.

[0106] In comparison, the methods that have been used to detect anti-HIVdrug resistance are less sensitive, time-consuming and technicallydemanding. The currently used methods include genotypic assays fordetecting HIV genome mutation based on PCR amplification of the viralRNA followed by sequencing of the amplified DNA templates, andphenotypic assays based on recombinant HIV virus (Hirsch, M. S. (1998)JAMA 279: 1964-1991). While the most sensitive PCR-based assay that hasbeen developed may not be sensitive enough to detect plasma HIV RNAbelow 50 copies/mL, false positivity for mutations may be generated dueto carry over from other HIV samples in the laboratory or from randompolymerase errors during PCR. The recombinant virus assay requires afirst RT-PCR amplification of plasma HIV RNA at more than 1000copies/mL, cloning the viral cDNA into an HIV vector, and then growingup the virus in permissive cell line. The whole process may take morethan two weeks to generate results and demand for highly skilledpersonnel to perform the test.

[0107] Thus, the methods provided in the present invention are moresensitive for detecting replicating HIV virus (at only about 5virions/mL), more efficient for testing for HIV drug resistance (lessthan a week), and more economic for high throughput screening.

[0108] 4. Methods for Designing Patient Customized HIV CocktailTreatments

[0109] Methods are also provided for taking a patient known to beinfected with one or more strains of the HIV virus and determining whatcombination of one or more anti-HIV agents will be effective in treatingthe patient. These methods can be used when a patient is initially beingtreated with anti-HIV agents or after a patient has been treated for aperiod of time with one or more anti-HIV agents and one or moreresistant strains may have developed resistance to the anti-H IV agentsbeing used.

[0110] In one embodiment, the method comprises:

[0111] taking a plurality of cell cultures, each of the culturescontaining recombinant cells (a) are capable of cell division, (b)express CD4 receptor and one or more additional cell surface receptorsnecessary to allow the HIV virus to infect, (c) enable the HIV virus toreplicate and infect the noninfected cells in the culture, and (d)comprises a reporter sequence introduced into the recombinant cellscomprising a reporter gene whose expression is regulated by a proteinspecific to HIV viruses which is expressed from a genome of an HIV virusupon infection of the recombinant cell by the HIV virus;

[0112] contacting the cell cultures with a sample containing the HIVvirus;

[0113] adding a different set of one or more anti-HIV agents to each ofthe cell cultures, either before or after contacting the cell cultureswith the sample; and

[0114] comparing expression of the reporter gene in the plurality ofcell cultures.

[0115] In one variation, each cell culture of the plurality is contactedwith a different set of one or more anti-HIV agents before beingcontacted with a sample containing the HIV virus.

[0116] In another variation, each cell culture of the plurality iscontacted with a different set of one or more anti-HIV drugs after beingcontacted with a sample containing the HIV virus and incubating for atime sufficient for the HIV virus replication to occur.

[0117] The anti-HIV agents can be any agents with known anti-HIVactivities, such as the ones described in Section 3, and combinationsthereof.

[0118] The culture of recombinant cells used in the method may be anycell which has the above described properties. The recombinant cellsdescribed in Section I are examples of cell having these properties andmay be used in this method.

[0119] Detecting a change in a level of expression of the reporter genein the cells in the culture may be performed by detecting a change in alevel of expression of the reporter gene in individual cells or a changein a level of expression of the reporter gene across the cell culture.

[0120] In one embodiment, detecting a change in a level of expressionincludes detecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

[0121] In yet another variation of this embodiment, the method furtherincludes comparing the change in the level of expression of the reportergene when different or no anti-HIV agents are used. For example, arecombinant cell culture that is contacted with the sample containingthe HIV virus but not with the one or more anti-HIV agents can serve asa negative control, while a recombinant cell culture that is contactedwith a sample containing HIV virus or a modified adenovirus, and the oneor more anti-HIV agents can serve as a positive control. By usingsuitable controls, inhibition of the reporter gene expression may bebetter correlated with anti-HIV efficacy of the agents.

[0122] It is noted that regulation of the reporter gene may be upregulation or down regulation. Accordingly, a change in the level ofexpression of the reporter gene may be an increase or decrease inreporter gene expression.

[0123] In one variation of this embodiment, the cell culture iscontacted with one or more anti-HIV agents before being contacted with asample containing the HIV virus. Alternatively, the cell culture may becontacted with one or more anti-HIV agents after being contacted with asample containing the HIV virus and incubating for a time sufficient forthe HIV virus replication to occur. Such preamplification of the HIVvirus may be advantageous for patient samples containing lower titer ofthe HIV virus to be tested against the anti-HIV agents.

[0124] The methods provided in this section can be used for screening ananti-HIV agent or agent combinations that are most active in inhibitingHIV viral infection and/or replication. The screening can be conductedagainst virtually all strains of HIV virus, regardless of theirgenotypes or tropisms. The results generated can help the physician ofHIV infected patients monitor HIV drug resistance, optimize the drugregimen and use the most efficacious drug “cocktail” to treat thepatient. By using such drug cocktails customized for each individualpatient and adjusted during the course of the treatment, physicians maysuccessfully prevent the HIV virus from developing drug resistance.Furthermore, physicians can avoid unnecessary side effects and drugtoxicity that would otherwise arise from treating a patient withineffective anti-HIV agents.

[0125] The ample and stable supply of the recombinant cells used inthese methods, as well as the ease of culturing the cells, enables oneto use the methods provided in this section in a high throughputscreening format to test many more drug cocktail combinations than wouldotherwise have been possible. Furthermore, because the HIV viruscontained in the sample from a patient may potentially harbor drugresistances strains, conventional drug screening may not have beeneffective in finding the optimum drug regimen. By using the methodsprovided in this section, the most efficacious drug regimen may bereadily identified by designing and testing exhaustive combinations ofdifferent drugs that target different components of the HIV virus or HIVreceptors.

[0126] 5. Methods for Screening Compositions for Anti-HIV Activity

[0127] The present invention also relates to methods for screeningcompositions which are not known to have anti-HIV activity for anti-HIVactivity. As used herein, a composition is intended to refer to anycomposition of matter, including single molecules, macromolecules suchas proteins and nucleotides, or combinations of two or more molecules ormacromolecules.

[0128] In one embodiment, the method comprises:

[0129] taking a culture of recombinant cells, which (a) are capable ofcell division, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in theculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus;

[0130] contacting the cell culture with a sample containing the HIVvirus;

[0131] adding one or more agents whose anti-HIV activity are unknown tothe cell culture either before or after contacting the cell cultureswith the sample; and

[0132] detecting a change in a level of expression of the reporter genein the cells in the culture.

[0133] The culture of recombinant cells used in the method may be anycell culture which has the above described properties. The recombinantcells described in Section I are examples of cells having theseproperties and may be used in this method.

[0134] The agents can be any anti-HIV drug candidates from naturalsources or synthetically generated. The agents can be any agenttargeting any components of the HIV virus, such as RT inhibitors,protease inhibitors, antisense and ribozyme oligonucleotides against HIVmRNA or viral RNA genome, decoys of TAR sequence or RRE (rev responseelement), competitive inhibitors like soluble CD4, Gag or Env proteinmutants, and agents that bind to HIV receptor or coreceptors and blockthe entry of HIV into the host cells.

[0135] Detecting a change in a level of expression of the reporter genein the cells in the culture may be performed by detecting a change in alevel of expression of the reporter gene in individual cells or a changein a level of expression of the reporter gene across the cell culture.

[0136] In one embodiment, detecting a change in a level of expressionincludes detecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

[0137] In another embodiment, detecting a change in a level ofexpression includes comparing a level of expression in a sample to alevel of expression in one or more control samples. For example, arecombinant cell culture that is contacted with a sample containing HIVvirus but not with any potentially anti-HIV agents can serve as anegative control, while a recombinant cell culture that is contactedwith a sample containing an HIV virus and the one or more agents thatare known to have anti-HIV activity can serve as a positive control. Byusing suitable controls, regulation of the reporter gene expression maybe better correlated with anti-HIV efficacy of the agents.

[0138] It is noted that regulation of the reporter gene may be upregulation or down regulation. Accordingly, a change in the level ofexpression of the reporter gene may be an increase or decrease inreporter gene expression.

[0139] In one variation of this embodiment, the cell culture iscontacted with one or more agents before being contacted with a samplecontaining the HIV virus. Alternatively, the cell culture may becontacted with one or more agents after being contacted with a samplecontaining the HIV virus and incubating for a time sufficient for theHIV virus replication to occur. This may be particularly advantageousfor the initial amplification of the HIV virus with low titer in thesample before being tested against the agents.

[0140] The methods described above can be used for high throughputscreening for anti-HIV drug candidates against various HIV containingsamples, especially for libraries of compounds generated bycombinatorial chemistry. These methods may be performed in any formatthat allows rapid preparation and processing of cells contained inmultiple-well plates, such as 96-well plates. Stock solutions of thetest agent as well as other assay reagents may be prepared manually andall subsequent pipetting, diluting, mixing, washing, incubating, samplereadout and data collecting may be done using commercially availablerobotic pipetting equipment, automated work stations, analyticalinstruments for detecting the signal generated by the assay. Examples ofsuch detectors include, but are not limited to, spectrophotometers,calorimeters, luminometers, fluorometers, and devices that measure thedecay of radioisotopes.

[0141] The methods described above are particularly cost-effective foruse in high throughput screening because the recombinant cells areimmortalized, easy to culture and more stable, compared to primary humancells such as PBMC cells. Furthermore, effects of multiple agents atmultiple doses on HIV infection and replication can be directlymonitored by detecting levels of reporter gene products in the 96-cellculture plates on a calorimetric or fluorescence plate reader.

[0142] 6. Constructing a Recombinant Cell Line According to the PresentInvention

[0143] The recombinant cells used in the present invention areimmortalized cells. Human tumor cell lines are preferably used. Othertransformed normal cells, such as human transformed primary embryonalkidney 293 cells, and human primary cells immortalized by transfectionwith telomerase (Bodnar, A.G. et al. (1998) Science 279:349-352) canalso be used.

[0144] In order to create a cell line which is permissive to HIVinfection, CD4 and one or more other HIV receptors are transfected,transduced or otherwise introduced into the immortalized cells. The oneor more other HIV receptors preferably include CXCR4 and CCR5 receptors.

[0145] CD4 receptor is believed to be the primary receptor for HIV entryinto the host cell. It has recently been discovered that specificchemokine receptors such as CXCR4 and CCR5 receptors play importantroles in mediating HIV entry and tropism for different target cells(reviewed by Berger, E. a. (1997) AIDS 11; Suppl. a: S3-S16; Dimitrov,D. S. (1997) Cell 91: 721-730). Macrophages-tropic (M-tropic) strains ofHIV virus can replicate in primary CD4⁺ T cells and macrophages and usethe beta-chemokine receptor CCR5 and less often, CCR3 receptor. T cellline-tropic (T-tropic) HIV strains can also replicate in primary CD4⁺ Tcells but can in addition infect established CD4⁺ T cell lines in vitrovia the alpha-chemokine receptor CXCR4. Many of the T-tropic strains canuse CCR5 in addition to CXCR4. Chemokine receptor like HIV coreceptorSTRL33 is expressed in activated peripheral blood lymphocytes and T-celllines and can function as an entry cofactor for Env proteins fromM-tropic, T-tropic and dual tropic strains of HIV-1 and SIV. Other HIVcoreceptors have also been identified by numerous in vitro assays,including chemokine receptors CCR2b, CCR3, CCR8 and CX3CR1 as well asseveral chemokine receptor-like orphan receptor proteins such asGPR15/BOB and STRL33/BONZO. Each or a set of these HIV coreceptors canmediate entry of different strains of HIV virus into the host cell. Bytransfecting, transducing or otherwise introducing these receptors intothe immortalized cell line, the host cell line can be renderedpermissive to HIV strains with broad-spectrum tropisms. In particular,by cell-cell fusion of the immortalized cell with cells expressing cellsurface receptors known to be involved in HIV infection such as T-cellsor monocytes, the immortalized cell can be transduced with various HIVreceptors simultaneously.

[0146] By transfecting, transducing or otherwise introducing a selectedset of coreceptors into an immortalized cell line or selectivelyexpressing certain coreceptors on the cell surface, a cell line can bedesigned which is permissive to certain strains of HIV and is not bepermissive to other strains of HIV. For example, CXCR4 coreceptor whichis required by T-tropic strains can be selectively expressed in therecombinant cells to allow infection of T-tropic strains of HIV.Meanwhile, M-tropic strains require CCR5 coreceptor to infect cells. Byhaving the recombinant cells not express CCR5 coreceptor, therecombinant cell line can selectively detect T-tropic strains in thepresence of M-tropic strains.

[0147] In order to detect HIV infection with a high level ofsensitivity, a “molecular switch” with high induction ratio isintroduced into the immortalized cell line expressing CD4 receptor andthe one or more additional HIV receptors. The molecular switch comprisesa reporter gene whose expression is induced when the cells are infectedby HIV. Various reporter genes can be used including lacZ (encodingβ-galactosidase), luciferases gene, CAT gene, SEAP gene, and genesencoding fluorescent proteins such as green fluorescent protein (GFP),enhanced blue fluorescent protein (EBFP), enhanced yellow fluorescentprotein (EYFP) and enhanced cyan fluorescent protein (ECFP).

[0148] The promoter region for the reporter gene contains a basicpromoter and a single or multiple copies of HIV specific enhancersequence. The basic promoter can be any cellular or viral basicpromoters such as the basic promoter regions of β-actin promoter,insulin promoter, human cytomegalovirus (CMV) promoter, HIV-LTR(HIV-long terminal repeat), Rous sarcoma virus RSV-LTR, and simian virusSV40 promoter. The HIV specific enhancer sequence can be any sequencethat can regulate the expression of the reporter gene via direct orindirect interaction with one or more HIV specific gene products. Forexample, the responsive element (TAR) for HIV transactivator protein Tatcan be used to enhance the expression of the reporter gene. Uponinfection of HIV, Tat expressed from the viral genome binds to TARsequence and, coupled with the basic promoter, induces expression of thereporter gene. More than one copy of TAR sequence can be linked tofurther enhance expression of the reporter gene and raise the inductionratios.

[0149] Alternatively, expression of the reporter gene can be induced byprotein-protein interactions between an HIV gene product, a DNA-bindingprotein (e.g. GAL4 DNA binding domain). a transactivator protein (e.g.VP16 transactivator domain derived from herpes simplex virus) that areexpressed by the host cell. Upon binding of the HIV specific geneproduct to the DNA binding protein as well as to the transactivatorprotein, reconstitution of a transcription factor is achieved bybringing the DNA-binding protein and the transactivator protein intoclose approximately. The reconstituted transcription factor can thenactivate downstream reporter gene expression via the specific bindingbetween the enhancer sequence (e.g. GAL4 enhancer sequence) upstream ofthe basic promoter with the DNA binding protein.

[0150] It should be noted that expression of a reporter gene can also beindirectly regulated by an HIV specific protein. For example,transcription of the reporter gene can be under the control a strongpromoter, such as the bacteriophage T7 or SP6 promoters, whileexpression of T7 or SP6 polymerase is regulated by a promoter comprisinga basic promoter and an HIV specific enhancer sequence. Upon binding ofthe HIV specific protein to the enhancer sequence, expression of T7 orSP6 polymerase is enhanced. As a result, T7 or SP6 polymerase expressedin the cell can then bind to the T7 or SP6 promoter upstream of thereporter gene and induce expression of the reporter gene in the cell.

[0151] Various methods can be used to introduce genes into theimmortalized cells. Examples of methods that may be used include, butare not limited to, calcium phosphate-mediated direction transfection,liposome-assisted transfection, and virus-mediated transfection. HIVreceptors can also be introduced into the host cell through cell fusionwith natural cells expressing these receptors on the cell surface.Clones of cells expressing the transfected genes may be selected byantibiotics such as hygromyin, G418, zeocin, etc., or based on herpessimplex virus tk gene. Expression of each receptor gene may be confirmedby Western blot to detect the protein with an antibody, Northern blot todetect the RNA with a nucleotide probe, or by FACS using the HIVreceptor expressed on the cell surface as antigens.

[0152] Two examples of plasmid vectors containing HIV receptor genes anda reporter gene are diagramed in FIGS. 1A and 1B.

[0153] As illustrated in FIG. 1A, CD4 and HIV co-receptors are expressedfrom SV40 early and late promoters in opposite directions. Genesencoding CD4 and CCR5 receptors are expressed from SV40 early promoterby a splicing mechanism at the SA sites. Genes encoding CXCR4 andhygromycin resistance are expressed bicistronically from SV40 latepromoter with Hygro being separated by an internal ribosome entry site(IRES). Expression of hygromycin resistance gene enables selection ofthe cell. The plasmid also contains prokaryotic replication origin andampicillin-resistance gene for DNA propagation in bacteria. The reportergene is carried by a separate plasmid that contains a second selectiongene (tk). The two plasmids may be co-transfected into HeLa cellssimultaneously or sequentially. Cell clones expressing all of thetransfected genes can be selected with antibiotics accordingly.

[0154] Genes encoding HIV receptor and coreceptors may also be expressedfrom the two retroviral vectors illustrated in FIG. 1B. The receptorsgene are expressed from the murine leukemia virus (MLV) LTR-promoter,each protein is expressed from a spliced mRNA or from an IRES (B.1). Thereporter sequence is carried by a second retroviral vector.Transcription of the reporter gene is in the opposite direction of theMLV LTR promoter with the enhancer sequence deleted in order to preventunregulated expression from the LTR promoter (B.2).

[0155] These vectors are packaged into infectious butreplication-incompetent virions by using a packaging cell line, such asthose stable or transient production lines based on the 293T cell line.The packaging cell line expresses all the necessary proteins, Gag, Poland Env, that are required for packaging, processing, reversetranscription, and integration of recombinant retroviral genomecontaining the Psi packaging signal.

[0156] The retroviral vectors are transfected into the packaging cellline. The virions produced in the packaging cells are then collected andused to infect a target cell. Since the virons arereplication-incompetent, the genes carried by the retroviral vectors arestably integrated into the target cell genome and can be expressed underthe control of the upstream promoter without producing infectiousvirions. The cells expressing all of the transduced genes can beselected with antibiotics and confirmed by Northern, Western blots orFACS accordingly. Alternatively, the cells expressing the reportersequence can be selected by infecting the cell culture with a modifiedadenovirus carrying HIV specific gene such as tat.

[0157] It should be noted that expression of HIV receptors can also becontrolled by an inducible promoter such as a tetracycline responsiveelement TRE. For example, one or more of the HIV coreceptors can beselectively presented on the cell surface by a controlled expressionusing the Tet-on and Tet-off expression systems provided by Clontech(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). In the Tet-on system, gene expression is activated by theaddition of a tetracycline derivative doxycycline (Dox), whereas in theTet-off system, gene expression is turned on by the withdrawn oftetracyline (Tc) or Dox. Any other inducible mammalian gene expressionsystems may also be used. Examples include systems using heat shockfactors, steroid hormones, heavy metal ions, phorbol ester andinterferons to conditionally expressing genes in mammalian cells.

[0158] Overall, the present invention provides novel recombinant celllines and methods using these cell lines. These methods are convenient,cost-effective and ultra sensitive for the detection of HIV infectionand replication. These methods can be very useful for high throughputscreening in preclinical drug discovery and development, as well asdesigning more efficacious anti-HIV drug cocktails in the clinic tocombat HIV drug resistance.

EXAMPLE

[0159] 1. Productive Infection of Recombinant HeLa Cells with HIV virus

[0160] A recombinant cell line was established from human cervicalcancer HeLa cells. The HeLa cells were cotransfected with an expressionvector (pRepD4R4) and a vector (pTAR3CIac) at a 1:1 ratio. As shown inFIG. 2A the expression vector pRepD4R4 includes CD4 receptor and CXCR4receptor genes that are separated by an IRES sequence. As shown FIG. 2Bthe vector pTAR3CIac includes a reporter sequence comprising a promoterregion that includes three copies of TAR sequences and a CMV basicpromoter, and a lacZ reporter gene whose expression is under the controlof the promoter. The stably-transfected cells were selected by culturingin medium containing G418 at 900 μg/ml. Each clone of the cells selectedwas subsequently cultured in duplicates, and one of the duplicates wasinfected with a low titer HIV stock solution. The low-titer HIV stockwas collected from supernatant of a HeLa cell culture that wastransfected with a B-cell tropic HIV provirus DNA (strain GRCSF) andincubated for 3 days post transfection.

[0161] Upon infection of HIV contained in the stock solution, Tatprotein expressed from the viral genome binds to TAR and inducesexpression of lacZ reporter gene to produce high level ofβ-galactosidase. The cell clones expressing β-galactosidase and stainedblue with X-gal were identified, and the cells from the uninfectedduplicate of the darkest blue colony were propagated. Such selectedcells were designated as HeLaD4R4 cells.

[0162] HeLaD4R4 cells constructed as described above were tested for HIVinfection. HeLaT4 cells (also called HT4) which express human CD4receptor were used as a control. The HeLaD4R4 cells and HeLaT4 cellswere grown up in DMEM and 5% bovine calf serum.

[0163] Exponentially growing cells were cultured in a six-well plate andinfected with 1 ml of a diluted HIV stock (about 10 infectiousparticles/ml) obtained from HIV provirus transfected HeLa cell cultureas described above. The cells were continuously cultured, and fixed with1% formaldehyde for 2 minutes 1, 3, 4, 5 days after the initialinfection. The cells were fixed with 0.5% formaldehyde for 2 min. andstained with X-gal (0.5%) at 37° C. over night. Since the lacZ reportergene product, β-galactosidase, converts the substrate from colorless todark blue, cells expressing β-galactosidase as a result of beinginfected with HIV appear distinctly blue.

[0164]FIG. 3A shows the control HeLaT4 cells after three days of beingexposed to HIV. As can be seen, almost all of the HeLa cells were notstained blue, with few cell stained faint blue. This indicates thatcells without HIV CXCR4 were poorly infected and the HIV virus did notreplicate within the cell culture.

[0165] FIGS. 3B-2E shows HeLaD4R4 after 1, 3, 4, and 5 days. As can beseen in FIG. 3B, infection can be readily detected after 1 day, as shownby the blue cells. As can be seen in FIGS. 3C and 3D respectively,progressively more cells were infected and stained blue after 3 and 4days. As can be seen in FIG. 3E, virtually all cells in the well wereinfected and stained dark blue after 5 days.

[0166] The results shown in FIGS. 3B-3E indicate that following initialinfection of a few cells by about ten HIV virions, HIV was able toundergo a productive infection, i.e. an infection of a cell which isfully permissive for virus replication and production of progeny virions(Stevenson, M. AIDS 11 Suppl. a: S25-S33). In addition, the infectedcells appear to retain normal morphology, i.e. remaining attached to thesubstrate of the culture plate instead of rounding up and detaching fromthe plate

[0167] The results shown in FIG. 3E are particularly significant becauseHIV virions initially added to the sample were able to replicate withinthe cell culture and spread to infect other cells that are not infectedoriginally (compare FIGS. 3B and 3E). This is in significant contrast toan increase of cells stained blue simply due to cell division.

[0168]FIG. 3F illustrates a further experiment where AZT (100 μg/ml) wasadded to inhibit HIV replication and infection. As can be seen in FIG.3F, after four days of incubation in the presence of AZT only a fewclusters of cells were infected and stained blue. The sparse clusters ofblue cells are most likely cells divided from the few cells that wereinitially infected by the HIV virions added to the well.

[0169] By comparing FIG. 2F to FIGS. 3B-3E, one can see that AZT waseffective as an anti-HIV agent since the expression of the reporter genewas significantly reduced due to the presence of AZT. This comparison ofthe results in FIG. 3F to FIGS. 3B-3E is an example of how the presentinvention can be used to detect HIV drug resistance and to screencompositions for anti-HIV activity.

[0170] 2. Method for HIV Diagnosis

[0171] An example is provided for detecting HIV in a sample. This methodcan be used to diagnose a patient infected with HIV. According to themethod, recombinant cells are seeded into a multiple well plate. a smallamount of serum from an individual to be tested is added to duplicatesof the wells. After two to four days incubation, the cells are processedand the results are analyzed depending on the type of reporter geneused. For example, when lacZ gene is used as the reporter gene for therecombinant cells, the cells are treated with a processing solutioncontaining the substrate X-Gal for β-galactosidase, low concentration offormaldehyde (1%) and glutaraldehyde the (0.1%) to gently fix the cellswhile not inactivating the reporter protein. When a green fluorescentprotein (GFP) gene is as the reporter gene for the recombinant cells,the cells are observed under an UV microscope directly. The presence ofcells emitting green fluorescence indicate that the cells may have beeninfected by HIV virus contained in the sample. By using GFP as areporter gene replication of HIV can be directed monitored any timeduring the incubation without fixing and processing cells to ensure thatenough HIV virus has been replicated within the culture.

[0172] The above-described diagnosis test can be used as an independenttest for HIV infected patients, or in conjunction with HIV drugresistance and other HIV diagnosis tests.

[0173] a positive control agent may be used to ensure that therecombinant cells are responsive to HIV infection. a defective commoncold virus strain carrying an HIV tat gene that encodes HIVtransactivator protein Tat may be used as a positive control agent. Thecommon cold virus is used as a vector to transfer the HIV tat gene intocells to mimic HIV infection. HIV itself may not be ideal for use as apositive control because HIV may not be sufficient stable and can easilylose its activity, thus the virus may not be stored for an extendedperiod of time. In contrast, the common cold virus can be dried intopowder and stored for a long time. In addition, this strains of commoncold virus is derived from a strain of common cold virus (adenovirustype 5) that is defective in viral replication, therefore safer for anextensive usage as a positive control.

[0174] 3. Method for Detecting HIV Drug Resistance

[0175] An example of how to perform the method for detecting HIV drugresistance is provided. Recombinant cells are seeded into each well of amultiple-well plate. Duplicate wells contain each anti-HIV agent to betested. a small amount of patient serum is added to each well andincubated for a few days. After two to four days of incubation, thecells are processed and the results are analyzed depending on the typeof reporter gene used. For example, when lacZ gene is used as thereporter gene for the recombinant cells, the cells are treated withprocessing solution containing the substrate X-Gal for β-galactosidase,low concentration of formaldehyde (1%) and glutaraldehyde the (0.1%) togently fix the cells while not inactivating the reporter protein. Forquantitative analysis, levels of beta-Gal can be measured by an ONPGassay on the cell extract. When a green fluorescent protein (GFP) geneis used as the reporter gene for the recombinant cells, the cells areobserved under an UV microscope directly. For quantitative analysis, thefluorescent cells are sorted by FACS and numbers of cells expressing theGFP reporter are measured.

[0176] If the cells in the wells containing a particular drug expressthe reporter gene at a sufficient level, it indicates that the HIV viruscontained in the sample may be resistant to the drug at the tested dose,and the virus has replicated and spread the infection among therecombinant cells in the presence of the anti-HIV drug.

[0177] Wells where no serum sample has been added can be used as anegative control. Negative controls can be performed for each agentbeing tested. a positive control, for example using the positive controlagent described in Example 2 (adenovirus carrying HIV tat gene), canalso be performed for each agent tested to ensure that the recombinantcells function properly.

[0178] 4. Method for Determining Viral Load in a Patient Serum

[0179] An example of how to perform the method for determining viralload in patient serum is provided. About 1 milliliter of patient's serumis diluted progressively, such as 1:10, 1:100, 1:1000, etc, and added towells containing the recombinant cells. The highest dilution that stillinduces expression of the reporter gene of the recombinant cells in thewell is the titer (concentration) of the HIV in the patient serum. Whenthe viral load become low, finer steps of dilution may be performed todetermine more accurately the numbers of viral particles in thepatient's serum.

[0180] The method can be used to determine how many viral particles permilliliter are present in patient serum. Since the recombinant cells ina culture are sensitive to infection of even a single virion, thismethod can detect infection by only one viral particle, thereforesuitable for detecting a patient sample containing low titer HIV, even afew viral particles per milliliter of patient serum. Such a highsensitivity is important for monitoring the progress of anti-HIV drugtreatment. Compared to the “ultra-sensitive” PCR-based assays that canonly detect hundreds or more viral particles per milliliter of patientserum, this method is more sensitive and can be used to detect muchlower titer HIV in the sample. This is particularly important fordetecting HIV virus in a patient sample after anti-HIV drug treatmentwhen viral titer is below the detectable level of conventional HIVdetection methods.

[0181] 5. Method for Screening for Anti-HIV Agents

[0182] Described here is an example of a method for performing highthroughput anti-HIV drug screening. To screen for new anti-HIV agents,the recombinant cells are seeded into a multiple well plate, such as96-well plate. To each well the agent to be tested for anti-HIV activityis added. a small amount of HIV stock is added to each well, so that thecells in each well are infected with about 10 viral particles. After afew days of incubation, the cells in the wells are analyzed on acalorimetric or fluorescence plate reader. The wells are compared withone or more wells containing the recombinant cell and virus but not theagent. Inhibition of the expression of the reporter gene in wellscontaining an agent indicates that the agent may have anti-HIV activityat the tested dose. Once potential anti-HIV agents have been identified,the test may be repeated to further confirm the anti-HIV activity of theagent.

[0183] Throughout this application, various publications are referenced.The disclosures of these publications, and the references cited therein,in their entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

[0184] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexample be considered as exemplary only, with a true scope and spirit ofthe invention being indicated by the claims.

What is claimed is:
 1. A recombinant cell comprising: a reportersequence introduced into the recombinant cells comprising a reportergene whose expression is regulated by a protein specific to HIV viruseswhich is expressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; the recombinant cell being capable ofcell division and expressing a CD4 receptor and one or more additionalcell surface receptors which facilitate productive infection of therecombinant cell by the HIV virus; and the recombinant cell enabling HIVvirus which has infected the recombinant cell to replicate and infectnon-infected cells in a culture of the recombinant cell.
 2. Therecombinant cell according to claim 1 wherein reporter gene expressionis up-regulated by the HIV specific protein.
 3. The recombinant cellaccording to claim 1 wherein reporter gene expression is down-regulatedby the HIV specific protein.
 4. The recombinant cell according to claim1 wherein the HIV specific protein is an HIV transactivator protein. 5.The recombinant cell according to claim 4 wherein the HIV transactivatorprotein is Tat.
 6. The recombinant cell according to claim 1 wherein theHIV specific protein is selected from the group consisting of HIVproteins Tat, Rev, Vpr, Vpx, Vif, Vpu, Nef, Gag, Env, RT, PR, and IN. 7.The recombinant cell according to claim 1 wherein the reporter sequencecomprises a promoter sequence including an HIV virus specific enhancersequence, and a reporter gene whose expression is regulated by bindingof an HIV specific transactivator protein to the HIV specific enhancersequence.
 8. The recombinant cell according to claim 7 wherein the HIVspecific transactivator protein is Tat and the HIV specific enhancersequence comprises at least one copy of TAR sequence.
 9. The recombinantcell according to claim 1 wherein the HIV specific protein regulatesexpression of the reporter sequence by a protein-protein interactionbetween the HIV specific protein and a transactivator protein present inthe recombinant cell.
 10. The recombinant cell according to claim 1wherein the reporter gene is selected from the group consisting ofβ-galactosidase, luciferase, beta-glucuronidase, chloramphenicol acetyltransferase (CAT), secreted embryonic alkaline phosphatase (SEAP),hormones and cytokines.
 11. The recombinant cell according to claim 1wherein the one or more additional cell surface receptors expressed bythe recombinant cell are selected from the group consisting of CXCR4,CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1,STRL33/BONZO and GPR15/BOB.
 12. The recombinant cell according to claim1 wherein the one or more additional cell surface receptors expressed bythe recombinant cell comprises CXCR4.
 13. The recombinant cell accordingto claim 1 wherein the one or more additional cell surface receptorsexpressed by the recombinant cell comprises CCR5.
 14. The recombinantcell according to claim 1 wherein the one or more additional cellsurface receptors expressed by the recombinant cell comprises CXCR4 andCCR5.
 15. The recombinant cell according to claim 1 wherein therecombinant cell expresses a sufficient number of cell surface receptorsto render the recombinant cell permissive to substantially all strainsof HIV.
 16. The recombinant cell according to claim 1 wherein therecombinant cell is a product of cell fusion with a cell which expressesone or more cell surface receptors selected from the group consisting ofCXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1,STRL33/BONZO and GPR15/BOB.
 17. The recombinant cell according to claim1 wherein the recombinant cell is a tumor cell.
 18. The recombinant cellaccording to claim 1 wherein the recombinant cell is a cell which hasbeen immortalized by introducing a gene into the cell which renders thecell line immortalized.
 19. A kit comprising: first and secondrecombinant cell lines, each recombinant cell line comprising: areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus, the recombinant cell linebeing capable of cell division and expressing a CD4 receptor and one ormore additional cell surface receptors which facilitate productiveinfection of the recombinant cell by the HIV virus, and the recombinantcell line enabling the HIV virus which has infected the recombinant cellto replicate and infect non-infected cells in a culture of therecombinant cell; wherein the one or more additional cell surfacereceptors which the first recombinant cell line expresses renders thefirst recombinant cell line permissive to a first group of strains ofHIV and the one or more additional cell surface receptors which thesecond recombinant cell line expresses renders the second recombinantcell line permissive to a second, different group of strains of HIV. 20.The kit according to claim 19 wherein the first and second recombinantcell lines are mixed together in the kit.
 21. The kit according to claim19 wherein the first recombinant cell line includes a first reportergene and the second recombinant cell line includes a second differentreporter gene which allows the first and second recombinant cell linesto be independently identified.
 22. The kit according to claim 21wherein the first and second recombinant cell lines are mixed togetherin the kit.
 23. A method for detecting a presence of HIV virus in asample comprising: taking a culture of recombinant cells which (a) arecapable of cell division, (b) express CD4 receptor and one or moreadditional cell surface receptors necessary to allow the HIV virus toinfect, (c) enable the HIV virus to replicate and infect the noninfectedcells in the cell culture, and (d) comprise a reporter sequenceintroduced into the recombinant cells comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; contacting the cell culture with asample to be analyzed for the presence of HIV virus in the sample; anddetecting a change in a level of expression of the reporter gene incells in the recombinant cell culture.
 24. A method for detecting thepresence of different strains of HIV virus in a sample comprising:taking a first culture of recombinant cells which (a) are capable ofcell division, (b) express CD4 receptor and one or more additional cellsurface receptors which render the first cell culture permissive to afirst group of strains of HIV but does not render the first cell culturepermissive to a second, different group of strains of HIV, (c) enablethe HIV virus to replicate and infect the noninfected cells in the cellculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; taking a second culture of recombinant cells which (a) arecapable of cell division, (b) express CD4 receptor and one or moreadditional cell surface receptors which render the second culturepermissive to the second group of strains of HIV but does not render thesecond cell culture permissive to the first group of strains of HIV, (c)enable the HIV virus to replicate and infect the noninfected cells inthe cell culture, and (d) comprise a reporter sequence introduced intothe recombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the first and second cell cultures with a sample to beanalyzed for the presence of different strains of HIV virus; detecting achange in a level of expression of the reporter gene in cells in thefirst cell culture; detecting a change in a level of expression of thereporter gene in cells in the second cell culture; and distinguishingbetween the first and second groups of strains based on whether a changein a level of expression of the reporter gene occurs in the first or thesecond cell culture.
 25. A method for detecting HIV drug resistance in asample comprising: taking a culture of recombinant cells which (a) arecapable of cell division, (b) express CD4 receptor and one or moreadditional cell surface receptors necessary to allow the HIV virus toinfect, (c) enable the HIV virus to replicate and infect the noninfectedcells in the cell culture, and (d) comprise a reporter sequenceintroduced into the recombinant cells comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; contacting the cell culture with asample containing HIV virus; adding one or more anti-HIV agents to thecell culture either before or after contacting the cell culture with thesample; and detecting a change in a level of expression of the reportergene in the cells.
 26. The method according to claim 25 wherein adifferent set of one or more anti-HIV agents is added to each cellculture of the plurality of cell cultures before the cell culture iscontacted with a sample containing the HIV virus.
 27. The methodaccording to claim 25 wherein a different set of one or more anti-HIVagents is added to each cell culture of the plurality of cell culturesbefore the cell culture is contacted with a sample containing the HIVvirus.
 28. The method according to claim 25 wherein the anti-HIV agentsinclude agents selected from the group consisting of nucleoside HIV RTinhibitors, nonnucleoside RT inhibitors, and protease inhibitors. 29.The method according to claim 25 wherein the anti-HIV agents includeagents selected from the group consisting of ZIDOVUDINE, DIDANOSINE,ZALCITABINE, LAMIVUDINE, STAVUDINE. ABACAVIR, NEVIRAPINE, DELAVIRDINE,EFAVIRENZ, INDINAVIR, RITONAVIR, SAQINAVIR, NELFINAVIR, and AMPRENAVIR.30. A method for taking a patient known to be infected with one or morestrains of the HIV virus and determining what combination of one or moreanti-HIV agents would be effective in treating the patient, the methodcomprising: taking a plurality of cell cultures, each of the culturescontaining recombinant cells which (a) are capable of cell division, (b)express CD4 receptor and one or more additional cell surface receptorsnecessary to allow the HIV virus to infect, (c) enable the HIV virus toreplicate and infect the noninfected cells in the cell culture, and (d)comprises a reporter sequence introduced into the recombinant cellscomprising a reporter gene whose expression is regulated by a proteinspecific to HIV viruses which is expressed from a genome of an HIV virusupon infection of the recombinant cell by the HIV virus; contacting thecell cultures with a sample containing the HIV virus; adding a differentset of one or more anti-HIV agents to each of the cell cultures eitherbefore or after contacting the cell cultures with the sample; andcomparing expression of the reporter gene in the plurality of cellcultures.
 31. The method according to claim 30 wherein a different setof one or more anti-HIV agents is added to each cell culture of theplurality of cell cultures before the cell culture is contacted with asample containing the HIV virus.
 32. The method according to claim 30wherein a different set of one or more anti-HIV agents is added to eachcell culture of the plurality of cell cultures after the cell culture iscontacted with a sample containing the HIV virus.
 33. The methodaccording to claim 30 wherein the anti-HIV agents include agentsselected from the group consisting of nucleoside HIV RT inhibitors,nonnucleoside RT inhibitors, and protease inhibitors.
 34. The methodaccording to claim 30 wherein the anti-HIV agents include agentsselected from the group consisting of ZIDOVUDINE, DIDANOSINE,ZALCITABINE, LAMIVUDINE, STAVUDINE, ABACAVIR, NEVIRAPINE, DELAVIRDINE,EFAVIRENZ, INDINAVIR, RITONAVIR, SAQINAVIR, NELFINAVIR, and AMPRENAVIR.35. A method for screening compositions for anti-HIV activitycomprising: taking a culture of recombinant cells which (a) are capableof cell division, (b) express CD4 receptor and one or more additionalcell surface receptors necessary to allow the HIV virus to infect, (c)enable the HIV virus to replicate and infect the noninfected cells inthe cell culture, and (d) comprise a reporter sequence introduced intothe recombinant cells comprising a reporter gene whose expression isregulated by a protein specificto HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the cell culture with the HIV virus; adding one ormore agents whose anti-HIV activities are unknown to the cell cultureeither before or after contacting the cell cultures with the HIV virus;and detecting a change in a level of expression of the reporter gene inthe cells in the culture.
 36. The method according to claim 35 whereinthe agents are added to the cell culture before the cell culture iscontacted with the HIV virus.
 37. The method according to claim 35wherein the agents are added to the cell culture after the cell cultureis contacted with the HIV virus.
 38. The method according to any one ofclaims 23-37 wherein the recombinant cell comprises: a reporter sequenceintroduced into the recombinant cells comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; the recombinant cell being capable ofcell division and expressing a CD4 receptor and one or more additionalcell surface receptors which facilitate productive infection of therecombinant cell by the HIV virus; and the recombinant cell enabling theHIV virus which has infected the recombinant cell to replicate andinfect non-infected cells in a culture of the recombinant cell.
 39. Themethod according to any one of claims 23-37 wherein the HIV specificprotein is selected from the group consisting of HIV proteins Tat, Rev,Vpr, Vpx, Vif, Vpu, Nef, Gag, Env, RT, PR, and IN.
 40. The methodaccording to any one of claims 23-37 wherein the HIV specific protein isan HIV transactivator protein.
 41. The method according to claim 40wherein the HIV transactivator protein is Tat.
 42. The method accordingto any one of claims 23-37 wherein the reporter sequence comprises apromoter sequence including an HIV virus specific enhancer sequence, anda reporter gene whose expression is regulated by binding of an HIVspecific transactivator protein to the HIV specific enhancer sequence.43. The method according to claim 42 wherein the HIV specifictransactivator protein is Tat and the HIV specific enhancer sequencecomprises at least one copy of TAR sequence.
 44. The method according toclaim 38 wherein the one or more additional cell surface receptorsexpressed by the recombinant cell are selected from the group consistingof CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3,CX₃CR1, STRL33/BONZO and GPR15/BOB.
 45. The method according to claim 38wherein the one or more additional cell surface receptors expressed bythe recombinant cell comprises CXCR4.
 46. The method according to claim38 wherein the one or more additional cell surface receptors expressedby the recombinant cell comprises CCR5.
 47. The method according toclaim 38 wherein the one or more additional cell surface receptorsexpressed by the recombinant cell comprises CXCR4 and CCR5.
 48. Themethod according to any one of claims 23-37 wherein detecting a changein a level of expression of the reporter gene in the cells includesdetecting a change in a level of expression of the reporter gene inindividual cells.
 49. The method according to any one of claims 23-37wherein detecting a change in a level of expression of the reporter genein the cells includes detecting a change in a level of expression of thereporter gene across the cell culture.
 50. The method according to anyone of claims 23-37 wherein detecting a change in a level of expressionof the reporter gene in the cells includes detecting whether viralreplication within the cell culture has occurred.
 51. The methodaccording to any one of claims 23-37 wherein detecting a change in alevel of expression of the reporter gene in the cells includes comparinga level of expression in cells contacted with the sample to a level ofexpression cells contacted with one or more control samples.
 52. Themethod according to any one of claims 23-37 wherein detecting a changein a level of expression of the reporter gene in the cells includesdetecting an increase in expression of the reporter gene.
 53. The methodaccording to any one of claims 23-37 wherein detecting a change in alevel of expression of the reporter gene in the cells includes detectinga decrease in expression of the reporter gene.
 54. The method accordingto any one of claims 23-37 wherein the HIV used in the method iscontained in fluid derived from the group consisting of whole blood,blood serum, isolated peripheral blood cells, and T cells.